1. Technical Field
The present invention relates to methods and markers for identification of pre-malignancy and malignancy states utilizing extrachromosomal and intrachromosomal gene amplification. Further the present invention relates to the identification of specific genes which undergo extrachromosomal gene amplification and therapeutic interventions relating to their utility as therapeutic targets.
2. Background Art
The diagnosis of malignant conditions is approached from multiple directions as for example tissue biopsies, serum levels of specific markers (PSA for prostate as an example), mammography and the like. However, most of these methods do not identify pre-malignant cells where early diagnosis can significantly increase treatment potential. Further the identification of a malignant condition does not necessarily identify an underlying genetic abnormality which can be corrected utilizing gene therapy or suggest other points of therapeutic intervention.
Chronic lymphocytic leukemia (CLL), is the commonest leukemia, making up 30% of all cases (O'Brien, et al. 1995), However, the cause of this disease is unknown. The leukemia primarily effects elderly males and is characterized by the accumulation of morphologically mature-appearing B1-lymphocytes in peripheral blood, marrow, spleen and lymph nodes (O'Brien, et al., 1995). Prognosis in CLL is approximately assessed by Rai staging (Table I) and patient survival varies from 2 years (Rai III and IV) to >10 years (Rai 0) (Rai, et al., 1975). However, with each stage there is considerable variation in survival and patients can be further stratified according to the lymphocyte doubling time (Montserrat, et al., 1986). Patients with a short lymphocyte doubling time (<12 months) have a poorer survival rate than those with a longer doubling time (Montserrat, et al., 1986). At the present time, this disease is incurable but remissions can be obtained with alkylating agents, e.g., chlorambucil, or nucleoside analogs, e.g., fludarabine, but relapse and the eventual development of drug resistance is usually observed (O'Brien, et al., 1995).
The normal cellular counterpart of the CLL cell is in the mantle zone of the lymphoid follicle, and, like CLL cells, these lymphocytes are CD5+ B cells and have high levels of bcl-2 (Schena, et al., 1992). It is presumed that a small fraction of CLL cells are proliferating stem cells, possibly located in the lymphoid tissue or marrow, but the majority of cells are non-proliferating and accumulate most likely through defects in apoptosis.
The term genomic instability summarizes a variety of genomic alterations which include the loss or gain of chromosomes as well as genetic changes at the level of single genes, such as rearrangements, translocations, amplifications, deletions and point mutations, and has been considered to be a major driving force of multistep carcinogenesis (Nowell, 1976; Pienta et al., 1989; Temin, 1998; Solomon et al., 1991). Genomic integrity is maintained by checkpoint mechanisms; when cells suffer damage imposed by exposure to genotoxic drugs or microtubule toxins, the cell cycle is halted until the damage is repaired or apoptosis is initiated (for reviews, see Hartwell, 1992; Weinert and Lydall, 1993; Hartwell and Kastan, 1994).
Gene amplification represents one form of genomic instability in mammalian cells, although it can also occur as part of a normal developmental program in insects, amphibia, and lower organisms (Santelli, et al., 1991; Delikadis, et. al., 1989; Start, et. al., 1984). With one published exception (Prody, et al., 1989), gene amplification has not been observed in normal diploid cells (Lucke-Huhle, et al., 1989; Wright, et al., 1990; Tlsty, et al., 1990) and its presence indicates that these cells are genomically unstable, immortalized, transformed and/or tumorigenic. In mammalian cells lines and tumors, gene amplification has been described after drug selection (Stark, et al., 1993; Huang, et al., 1994; Huang, et al., 1994; Shah, et al., 1986), DNA damage (Lucke-Huhle, et al., 1989; Lucke-Huhle, et al., 1990; Yalkinoglu, et al., 1991) and as a result of c-Myc overexpression (Mai, et al., 1994; Denis, et al., 1991). Spontaneous gene amplification has also been reported (Johnston, et al., 1983). Gene amplification can occur in the presence of wildtype p53, but is facilitated by its absence (Yin, et al., 1992; Livingstone, et al., 1992); thus, gene amplification can involve both p53-dependent and -independent pathways (Van Der Bliek, et al., 1986; Zhou, et al., 1996). Gene amplifications often involves oncogenes, and more than 90% of these cases in patients involve c-myc where the degree of amplification correlates with the aggressiveness of tumor growth and poor prognosis (Schwab, et al., 1990).
c-Myc is a key regulator of growth, proliferation, differentiation, and development. Deregulation of the c-Myc oncoprotein has been reported in apoptosis, transformation, and in malignancies of lymphoid and non-lymphoid origin (Marcu, et al., 1992; Cole, et al., 1986). c-Myc plays a role in the modulation (Benevisty, et al., 1992; Bello-Fernandez, et al., 1993; Gaubatz, et al., 1994; Jansen-Durr, et al., 1993; Daksis, et al., 1994; Philipp, et al., 1994; Galaktinov, et al., 1996) and initiation of transcription (Roy, et al., 1993, Li, et al., 1994; Mai, et al., 1995). It is a short-lived nuclear oncoprotein (Cole, et al., 1986), which is strictly regulated during the cell cycle of normal diploid cells (Cole, et al., 1986; Heikkila, et al., 1987; Karn, et al., 1989). Increased half life of the protein is associated with immortalization and transformation (Marcu, et al., 1992; Cole, et al., 1986). The deregulation of c-Myc is a common feature in many tumors (Marcu, et al., 1992; Cole, et al., 1986), where it frequently is translocated (Stanton, et al., 1983; Potter, et al., 1992; Mai, et al., 1995; Marcu, et al., 1992; Cole, et al., 1986) and/or amplified and overexpressed (Marcu, et al., 1992; Cole, et al., 1986; Feo, et al., 1994; Alitalo, et al., 1985). In addition, the c-myc gene is often the site of proviral insertion (Marcu, et al, 1982; Cole, et al., 1986). Chromosomal aberrations involving c-myc are associated with a poor prognosis (Yokota, et al., 1986).
An amplified gene sequence is termed an “amplicon” and can be chromosomal (“homogeneously staining regions”, HSR) or extrachromosomal (“extrachromosomal elements”, Ees). Extrachromosomal submicroscopic amplicons that replicate are termed “episomes” (250–5,000 kb), and these can increase in size to be visible by light microscopy, at which point they are termed “double minutes” (>5,000 kb) (Stark, et al., 1993; Hahn, et al., 1993). A variety of mechanisms are involved in the production of gene amplification, and it appears likely that different mechanisms can be involved for different genes in the same cell or for the same gene in different cell types (Stark, et al., 1993; Stark, et al., 1989). The “replication models” predict that a localized replication even can allow an isolated part of the chromosome to repeatedly replicate, i.e., onion-skin, double rolling circle or chromosome-spiral models, and these amplified areas can remain intrachromosomal or be released extrachromosomally. The second major group of mechanisms are the “segregation-driven” mechanisms, i.e., deletion-plus-episome and sister chromatid exchange models. The deletion-plus-episome theory predicts that deletion of a portion of chromosome produces Ees which can proliferate and be subsequently incorporated into random sites on a variety of chromosomes (Carroll, et al., 1988; Windle, et al., 1991).
A model of cyclin D2 gene amplification in CLL (FIG. 4A) is an illustration of the dynamic nature of cyclin D2 gene amplification and is based on the data summarized in FIGS. 1–4. Cyclin D2 (on chromosome 12q13) can be amplified on chromosome 12 and thus give rise to an HSR. In addition, or alternatively, cyclin D2 can be found on extrachromosomal elements (Ees). The latter can be directly generated from the original locus. During this process, it is possible (but not obligatory) that one allele of cyclin D2 is detected. The extrachromosomal elements can re-integrate into chromosome 12q13 or into random loci on chromosome 12 or on other chromosomes. Alternatively, or additionally, Ees can remain extrachromosomally, but they can only be maintained as extrachromosomal structures if they contain replication origins. According to the EM studies, the Ees in CLL cells appear to propagate by replication (see FIGS. 4A and 4B). The dosage of cyclin D2 can also be increased due to the duplication of chromosome 12. Trisomy 12 is a frequently acquired aberration that occurs in a fraction of CLL patients (Crossen, et al., 1997; Dohner, et al., 1997).
Hamkalo et al, 1985 first showed using electron microscopy that the dihydrofolate reductase (DHFR) containing Ees in methorexate-resistant murine 3T3 cells are in circles, and numerous loops can be organized together in a rosette like structure. Similar findings have been observed by others in a variety of cell lines (Esnault, et al, 1994; Nonet, et, al., 1993; Sen, et al., 1994; Schneider, et al., 1992; Cohen, et al., 1996; Cohen, et al., 1997). Esnault et al (Esnault, et al., 1994) isolated 630 kb Ees from a methorexate-resistant cell line and demonstrated that each of these Ees contained on DHFR gene. Transfection of the Ees into the methotrexate-sensitive parent cell line can confer methotrexate resistance. The adenosine deaminase containing Ees in cells grown in 2′-deoxycoformycin (Nonet, et al., 1993), c-myc containing Ees in HL-60 cells (Sen, et al., 1994) and N-myc containing Ees from neuroblastoma cells (Schneider, et al., 1992) have been isolated and cloned. In general, the genes remain intact, can be in a high-to-tail or head-to-head configuration (perhaps depending on the duration of time the Ees have existed) and appear to contain additional genomic loci to the studied gene. It has been suggested that these Ees can replicate and different sized molecules can develop through intra-or inter-molecular recombination (Esnault, et al., 1994). In addition, the number of Ees can increase in replicating cells, if they are unequally segregated to each daughter cell and provide the cell with a survival advantage (Stark, et al., 1993; Hahn, et al., 1993).
CLL has been studied by comparative genomic hybridization, which detects areas of chromosomal gene amplification or deletions (Bentz, et al., 1995). Abnormalities are detected in 70% of patients and one-third of these will have amplifications of all or part of chromosome 12 (Bentz, et al., 1995). Amplifications at 12q23–24, 12q13–22 and 12q13–15 have been observed by FISH in one patient with CLL (Merup, et al., 1997). To date, extrachromosomal gene amplification has only rarely been observed in CLL, and one patient has been described with extrachromosomal amplification of c-myc (Wang, et al., 1991). Recently, it was demonstrated that amplification of the cyclin D2 gene is a constant finding in all CLL cells, and appears to be extrachromosomal and cyclin D2 sequences are also randomly integrated into multiple chromosomes (FIG. 2). The cause for the amplification is unknown, although by an extrapolation from the present murine studies, it can occur following prolonged m-Myc overexpression in the stem cells. As c-Myc mRNA is not increased in peripheral blood CLL cells (Greil, et al., 1991), the increase can occur in the CLL stem cells in lymphoid tissue or marrow; the cyclin D2 amplification can then persist in the circulating non-proliferating CLL cell, and, if they have replicative capacity, can actually increase in size and number in these cells.
Cyclin D2 is one of three D cyclins which can have an integral role in the cell cycle. The D cyclins increase during G1 and bind to cyclin dependent kinase-4 (CDK4) or CDK6 with the resulting phosphorylation of the retinoblastoma (Rb) protein and the release of the E2F transcription factors (Sherr, 1994; Hirama, et al., 1995). These factors can then induce the transcription of a variety of genes, e.g., c-myc, DHFR and myb, which area involved in DNA synthesis. It is likely that the three D cyclins have equivalent activities, although their predominant expression depends on the cell type, with cyclins D2 and D3 being primarily present in lymphoid tissue.
While there are some protocols available for providing prognosis but within each stage there is considerable variation in survival. Therefore a better protocol for providing a prognosis and for making decisions on therapeutic strategies is needed.